Christopher J. Phiel, Ph.D. Embryonic stem cells (ESCs) are unique among mammalian cells due to their ability to differentiate into any cell type, a process termed pluripotency. In order to remain pluripotent, ESCs must also resist external cues that promote cellular differentiation. Given the importance of ESCs to embryonic development, as well as for the development of future regenerative therapies, fully understanding the molecular basis of how ESCs retain their pluripotent state is of keen interest. Several years ago, it was shown that inhibition of the serine/threonine kinase, glycogen synthase kinase-3 (Gsk-3), promotes ESC pluripotency, yet the precise role for Gsk-3 inhibition in this context has been difficult to elucidate. Another cellular process that has been shown to regulate ESC pluripotency is reduced mRNA methylation (referred to as m6A). In our previously awarded grant, we established a novel mechanism for the regulation of m6A mRNA - Gsk-3 directly phosphorylates the RNA demethylase FTO, targeting FTO for subsequent ubiquitination and degradation via the proteasome. In the absence of Gsk-3? and Gsk-3? (Gsk-3 double knockout; DKO) in mouse ESCs, FTO phosphorylation and ubiquitination are impaired, leading to increased FTO levels and a concomitant 50% decrease in the amount of m6A mRNA. Taken together, our data directly link two seemingly unrelated aspects of ESC pluripotency; however, many details about the regulation of mRNA methylation remain to be investigated. Here, we propose to investigate three prominent questions. 1.) It is unknown whether the activation of signaling pathways upstream of Gsk-3 result in subsequent changes in mRNA methylation. We intend to investigate whether Wnt or phosphatidylinositol-3 kinase (PI3K) signaling, both known to regulate ESC pluripotency and both known to inhibit Gsk-3 activity, also leads to changes in mRNA methylation. In addition, it was recently shown that signaling via Smad2/3 down-regulates Mettl3, the RNA methyltransferase that promotes m6A. We will test whether a decrease in Mettl3 via Smad2/3 activation, in combination with increased FTO via Gsk-3 deletion or inhibition, more efficiently pushes ESCs to pluripotency. 2.) Unexpectedly, it was recently shown that elevation of the Krebs cycle intermediate ?- ketoglutarate, in the presence of Gsk-3 and mitogen-activated protein kinase kinase (MEK) inhibitors, enhanced the pluripotency of ESCs. Interestingly, ?-ketoglutarate is also required as a co-factor for FTO. Therefore, we intend to examine whether ?-ketoglutarate promotes pluripotency via reducing mRNA methylation. 3.) It has been reported that FTO preferentially demethylates dimethyladenosine (m6Am), yet we have not specifically investigated whether there are any m6Am changes in Gsk-3 DKO ESCs. Along with a colleague at the University of Colorado School of Medicine who has been using m6A individual-nucleotide- resolution cross-linking and immunoprecipitation (miCLIP), we intend to examine m6Am in wild-type (WT) and Gsk-3 DKO ESCs. This project is designed to allow the meaningful participation of undergraduates. Understanding how Gsk-3 intersects with mRNA methylation and cellular metabolism should provide a more complete picture of the fundamental mechanisms underlying embryonic stem cell pluripotency.